Vin AP 1,7, Zhai YG 2, Pang YG 5,9, Hu HB 6, Qiao L 2, Stubbs EB Jr 3,8, Perlman JI 1,4,7, Bu P 1,9 Department of Ophthalmology 1, Microbiology and Immunology 2, Neurology 3, and Pathology 4 Loyola University Chicago, Maywood, IL. Department of Biomedical Engineering 5 at Illinois Institute of Technology, Chicago, IL. Department of Medicine 6, University of Florida, Malcolm Randall VA Medical Center, Gainesville, FL. Surgery 7, Neurology 8, and Research 9 Edward Hines, Jr. VA Hospital, Hines, IL. Retinal ischemia is a common cause of visual impairment and blindness among individuals over 50 years of age in the United States. Ischemic insult to the human retina can be observed in open-angle glaucoma, diabetic retinopathies, and hypertensive retinopathies. Current therapies for retinal ischemic disease are not satisfactory and have little impact on preventing or slowing the molecular process that leads to retinal ganglion cell death. The mechanism of cell death induced by retinal ischemia is not completely understood. It is known that ischemic retinal injury leads to energy dependent dysfunction, tissue edema, and eventual retinal ganglion cell death (1). Ischemia-induced neuronal injury is associated with enhanced production of endogenous substances such as glutamate, oxygen free radicals, nitric oxide (NO) and calcium (2, 3, 4). Interestingly, glutamate in particular acts as a normal neurotransmitter in the retina, but at high levels is neurotoxic in vitro and in vivo, resulting in apoptosis of retinal ganglion cells. The major causes of cell death from glutamate are the influx of calcium into cells and the generation of free radicals (5). Resveratrol (trans-3, 5, 4’-trihydroxystilbene) is a polyphenol which is present at high levels in the skin and seeds of grapes, nuts, and pomegranates. It also constitutes one of the major components of red wine. Resveratrol has been reported to have anti-inflammatory and anti-aging, antioxidant, and anti-tumor activities as well as important protective effects in the nervous system. In a recent study, resveratrol was found to protect the spinal cord, kidneys, and heart from ischemia-reperfusion injury through upregulation of NO (6). The role of resveratrol on retinal neurons during or after ischemia is unknown. In this study, we aimed to determine whether resveratrol has protective effects on retinal cells during simulated acute retinal ischemia in vitro as well as in vivo. INTRODUCTION In vitro study: Confluent cultures of R28 cells were treated with L-glutamate (0-6 mM) for 24 hours in the absence or presence of resveratrol (1-2 µg/ml). Cell viability after treatment was determined with a neutral red dye uptake assay. In vivo study: Retinal ischemia was induced in adult male Sprague Dawley rats by intracameral elevation of intraocular pressure for 45 minutes. Following ischemic insult, rats received (intraperitoneally) an equal volume of vehicle (20% DMSO) or resveratrol (20 mg/kg) daily for 6 days. Retinal function was determined by relative changes in electroretinographic (ERG) responses from dark-adapted rats assessed prior to and 7 days after ischemic insult. METHODS 1. Hughes, W. F. (1991). Quantitation of ischemic damage in the rat retina. Exp Eye Res 53, Choi, D. W. (1995). Calcium: still center-stage in hypoxic-ischemic neuronal death. Trends Neurosci 18, Dreyer, E. B. (1998). A proposed role for excitotoxicity in glaucoma. J Glaucoma 7, Louzada-Junior, P., Dias, J. J., et al. (1992). Glutamate release in experimental ischaemia of the retina: an approach using microdialysis. J Neurochem 59, Aoun, P, Simpkins J.W., Agarwal, N. (2003). Role of PPAR-gamma ligands in neuroprotection against glutamate- induced cytotoxicity in retinal ganglion cells. IOVS 44, Li, C., Yan, Z., et al. (2010). Neuroprotective effects of resveratrol on ischemic injury mediated by modulating the release of neurotransmitter and neuromodulator in rats. Neurochem Int 56, Mokni, M., Limam, F., et al. (2007). Strong cardioprotective effect of resveratrol, a red wine polyphenol, on isolated rat hearts after ischemia/reperfusion injury. Arch Biochem Biophys 457, Giovannini, L., Migliori, M., et al. (2001). Resveratrol, a polyphenol found in wine, reduces ischemia reperfusion injury in rat kidneys. J Cardiovasc Pharmacol 37, Huang, S. S., Tsai, M. C., et al. (2001). Resveratrol reduction of infarct size in Long-Evans rats subjected to focal cerebral ischemia. Life Sci 69, Tsai, S. K., Hung, L. M., et al. (2007). Resveratrol neuroprotective effects during focal cerebral ischemia injury via nitric oxide mechanism in rats. J Vasc Surg 46, Anekonda, T. S., and Adamus, G. (2008). Resveratrol prevents antibody-induced apoptotic death of retinal cells through upregulation of Sirt1 and Ku70. BMC Res Notes 1, 122. REFERENCES Role of Resveratrol in Neuroprotection Against Glutamate-Induced Cytotoxicity in Retinal Precursor Cells Acknowledgements: This work was supported by The Richard A. Perritt Charitable Foundation and Illinois Society for the Prevention of Blindness (ISPB). Resveratrol protects cultured R28 cells against glutamate-induced cell cytotoxicity in vitro. In vivo, resveratrol-treated rats showed improvement in ERG a-wave and b-wave amplitudes following ischemic insult as compared to vehicle-treated rats. These preliminary findings suggest that resveratrol has therapeutic value in the management of retinal ischemic diseases. CONCLUSIONS The major finding of this preliminary study is that resveratrol has a statistically significant protective effect against glutamate-induced cytotoxicity in vitro. In vivo, there appears to be a trend towards improved a-wave and b-wave amplitudes with use of resveratrol as compared to inert vehicle, although only a-wave amplitudes were shown to be significantly better statistically when comparing the two treatment groups. Recent studies have shown that resveratrol protects tissues of the heart, kidney, and brain from ischemia / reperfusion injury (7,8,9). Resveratrol significantly protected rat brain tissue against focal cerebral ischemia (10). A recent study also showed that resveratrol has a preventative effect in antibody-induced apoptotic death of retinal cells (11). There is much left to discover in exploring the use of resveratrol in ischemic retinal disease. The exact mechanism of action of resveratrol such as observed in this study remains unknown. Studies have suggested that a mechanism along the nitric oxide pathway may play a role (6). The range of therapeutic levels of resveratrol as well as the minimum systemic concentration required to reach therapeutic levels in the retina have not yet been elucidated. Furthermore, while in our small sample size, the a-wave and b-wave amplitudes appeared to be somewhat better in the resveratrol-treated group versus the vehicle-treated group, the subjective functional significance of this improvement is unclear. Further studies with larger sample sizes are indicated to explore these questions further. Our preliminary findings raise awareness for the potential of resveratrol to protect against ischemic retinal disease-related retinal ganglion cell death. DISCUSSION Figure 3. Cell survival with resveratrol in the presence of glutamate. Results of neutral red dye uptake assay on R28 cells (10,000 cells per well) treated with 6mM glutamate and either 1μg/mL or 2μg/mL resveratrol. * p < Student’s t-test (n = 6 per group) comparing cells + glutamate only data to cells + glutamate + resveratrol data. Resveratrol at concentrations of 1μg/mL or 2μg/mL has a statistically significant protective effect on survival of R28 cells (at 10,000 cells per well) treated with 6mM glutamate. Figure 2. Effect of glutamate concentration on cell survival. Results of neutral red dye uptake assay on R28 cells (10,000 cells per well) treated with increasing doses of glutamate. Statistically significant changes in cell survival were observed at glutamate concentrations of 5mM and 6mM. * p<0.001, Student’s t-test (n = 6 per group) comparing survival data between control and each glutamate treatment group. RESULTS Figure 1. Effect of glutamate and resveratrol on the morphology of R28 cells. Shown are representative photographs of R28 cells (20,000 cells per well) treated with glutamate 2.5mM or glutamate 2.5mM + resveratrol 5μg/mL. Control + Glutamate + Resveratrol Control + GlutamateControl Cells Effect of Resveratrol Treatment on Retinal Function Following Ischemic Insult Pre-ischemia Post-ischemia VehicleResveratrol Vehicle Resveratrol Pre Post a-waveb-wave Scotopic dB Flash Amplitude (μV) Figure 4. Effect of resveratrol treatment on retinal function following ischemic insult. (Upper panels) ERG recordings from Sprague Dawley rats treated with vehicle (20% DMSO) or resveratrol (20 mg/kg) as indicated. Tracings shown were obtained prior to (pre-ischemia) and 1-week (post-ischemia) following ischemic insult. (Lower panels) Quantitative changes in ERG a- and b-wave amplitudes as indicated. Pre, prior to ischemic insult; Post, 1-week following ischemic insult. * p<0.05, Student’s t-test (n = 4 per group) comparing post-ischemic insult data between vehicle- and resveratrol-treated rats. *